1. Field of the Invention
The present invention relates generally to the fields of histology and molecular biology. More particularly, it concerns compositions and methods for preparing a tissue section that allows RNA in the section to be recovered more abundantly and in a more intact form than previous methods in which significant degradation occurred.
2. Description of Related Art
Histological staining of thin tissue sections mounted on slides is frequently used to improve the ability to distinguish specific subregions and cellular structures during microscopic examination. Different stains, optimized for visualization of different structures in different types of tissue, for example liver, brain, kidney, tumor biopsies, etc. are used by pathologists and researchers for specific applications, for example diagnosis of metastatic disease based on abnormal tissue morphology.
In addition to microscopic examination, molecular tests are increasingly being carried out on thin tissue sections in order to provide additional information about the sample, including detection of mutations and assessment of patterns of gene expression. Molecular tests require extraction of nucleic acid, i.e., DNA and RNA, from the sectioned tissue.
Molecular tests based on RNA analysis provide information about the expression of genes in the tissue sample, for example quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is one type of molecular assay that is used to determine whether and to what extent given genes are expressed in a tissue sample. Microarray expression profiling is another example of an RNA-based molecular test that can be used to determine which subset of genes are expressed in a specific tissue sample.
RNA is a particularly labile biomolecule, being much more susceptible to degradation by endogenous and exogenous nucleases and to nonspecific degradation by divalent cations, heat, and elevations in pH, compared to DNA. Extraction of RNA from tissue sections in an intact form is required for subsequent T7 RNA polymerase-mediated linear amplification using various strategies (ref Eberwine patents and papers). Linear amplification is generally required for microarray analysis on thin tissue sections, since the amount of RNA needed for conversion to cDNA in order to carry out microarray hybridization is several micrograms, and this amount of RNA cannot generally be obtained from this type of sample.
Treatment of tissue with aldehyde fixatives such as formalin and paraformaldehyde causes chemical crosslinking of nucleic acid, which compromises the ability to extract intact RNA and/or to carry out reverse transcription using RNA from the fixed samples. For this reason, the alternative processing method using frozen tissue, rather than aldehyde-fixed tissue, is recommended in cases where the RNA will be extracted for molecular tests, especially for microarray expression profiling. Successful extraction of intact RNA from frozen sections is however also challenging, because the endogenous nucleases in the tissue, especially ribonucleases, must be maintained in an inactive form during the isolation process. Whereas aldehyde-based fixatives generally result in irreversible inactivation of endogenous RNases (due to chemical crosslinking), the alternative tissue processing method, i.e., using frozen tissue, does not render endogenous ribonucleases permanently inactive. When the tissue is thawed, RNases generally become active. Endogenous RNase activity can result in partial or complete degradation of the tissue RNA, rendering it useless for molecular tests.
Methods used to inactivate endogenous RNases during sample processing include addition of placental RNase inhibitor to the reagents used for fixing and staining the tissue, and minimizing the time in which the tissue sections are exposed to aqueous environments. It is recognized that placing the tissue sections in aqueous solutions provides an opportunity for the endogenous RNases, which are inactive while in the frozen state, to become re-activated and degrade the cellular RNA.
However, protocols for fixing and staining frozen tissue sections generally include 3 main steps in which the sections are placed in aqueous solution (i.e., water). The steps commonly used to stain and process frozen tissue for histological examination are summarized as follows:                1. Sections are cut on a cryostat and transferred from the cryostat blade to clean glass microscope slides, where they are allowed to partially thaw for a few seconds in order to facilitate their adherence to the glass surface of the slide.        2. Sections mounted on slides are then “fixed” by submerging the slides successively into solutions of “graded ethanols”. Typically the graded ethanol series includes an initial submersion in 100% or 95% ethanol, followed by successive submersions in 75% or 70% ethanol, and finally in 50% ethanol. The suggested length of time in which the tissue is kept in each solution generally ranges from a few seconds to a few minutes.        3. After the graded ethanol series, the fixed tissue section is submerged in water for a period of several seconds to several minutes.        4. The tissue section is then submerged in the stain solution for a period of several seconds to several minutes. Examples of common histological stains are hematoxylin, eosin, and cresyl violet. The stains are typically either purchased as ready-to-use aqueous solutions, or are prepared by dissolving powdered stains in water.        5. The stained sections are then typically briefly submerged in water to remove excess stain. In some cases the sections may be stained with a second stain (“counter-stained”) to improve the ability to visualize certain cellular substructures.        6. The sections are then typically submerged in a second series of graded ethanol solutions of increasing ethanol concentration, for example, they may be placed sequentially into solutions of 50% ethanol, 70-75% ethanol, 95% ethanol, and 100% ethanol. This series serves to dehydrate the tissue section.        7. Certain applications such as laser capture microdissection (LCM, described in detail below), require that the stained sections be completely dry. To achieve this, the sections are transferred from 100% ethanol into xylene and in some cases followed by transferring to a second solution of xylene, in order to remove all residual ethanol from the section.        
A feature of many, if not all, of the published protocols for processing tissue is that they specify several steps in which the slides are dipped in water and aqueous staining solutions (see e.g., Kohda et al., 2000; Wong et al., 2000; Kazumori et al., 2001; Luzzi et al., 2001; Tanji et al., 2001). One paper stated that exposure to aqueous solutions destroys 99% of the mRNA (Murakami et al., 2000). The consensus from the literature is that RNA quality is improved in LCM samples by minimizing the time in which the tissue is exposed to aqueous environments, leading to recommendations that staining and destaining steps be reduced from minutes to seconds (Goldsworthy et al., 1999; Kohda et al., 2000; Kazumori et al., 2001; Tanji et al., 2001). However, RNA quality was not always improved simply by reducing the time the tissue sections were incubated in aqueous solutions.
Existing protocols continue to involve steps in which tissue samples are exposed to aqueous environments in which RNA can be degraded. Therefore, there is still a need for improved methods and compositions for preparing samples with RNA remaining intact.